Targeted single molecule sequencing methodology for ovarian hyperstimulation syndrome.

BACKGROUND: One of the most significant issues surrounding next generation sequencing is the cost and the difficulty assembling short read lengths. Targeted capture enrichment of longer fragments using single molecule sequencing (SMS) is expected to improve both sequence assembly and base-call accuracy but, at present, there are very few examples of successful application of these technologic advances in translational research and clinical testing. We developed a targeted single molecule sequencing (T-SMS) panel for genes implicated in ovarian response to controlled ovarian hyperstimulation (COH) for infertility. RESULTS: Target enrichment was carried out using droplet-base multiplex polymerase chain reaction (PCR) technology (RainDance®) designed to yield amplicons averaging 1 kb fragment size from candidate 44 loci (99.8% unique base-pair coverage). The total targeted sequence was 3.18 Mb per sample. SMS was carried out using single molecule, real-time DNA sequencing (SMRT® Pacific Biosciences®), average raw read length = 1178 nucleotides, 5% of the amplicons \u3e6000 nucleotides). After filtering with circular consensus (CCS) reads, the mean read length was 3200 nucleotides (97% CCS accuracy). Primary data analyses, alignment and filtering utilized the Pacific Biosciences® SMRT portal. Secondary analysis was conducted using the Genome Analysis Toolkit for SNP discovery l and wANNOVAR for functional analysis of variants. Filtered functional variants 18 of 19 (94.7%) were further confirmed using conventional Sanger sequencing. CCS reads were able to accurately detect zygosity. Coverage within GC rich regions (i.e.VEGFR; 72% GC rich) was achieved by capturing long genomic DNA (gDNA) fragments and reading into regions that flank the capture regions. As proof of concept, a non-synonymous LHCGR variant captured in two severe OHSS cases, and verified by conventional sequencing. CONCLUSIONS: Combining emulsion PCR-generated 1 kb amplicons and SMRT DNA sequencing permitted greater depth of coverage for T-SMS and facilitated easier sequence assembly. To the best of our knowledge, this is the first report combining emulsion PCR and T-SMS for long reads using human DNA samples, and NGS panel designed for biomarker discovery in OHSS

Prevalence, underlying causes, and preventability of sepsis-associated mortality in US acute care hospitals

Importance: Sepsis is present in many hospitalizations that culminate in death. The contribution of sepsis to these deaths, and the extent to which they are preventable, is unknown. Objective: To estimate the prevalence, underlying causes, and preventability of sepsis-associated mortality in acute care hospitals. Design, Setting, and Participants: Cohort study in which a retrospective medical record review was conducted of 568 randomly selected adults admitted to 6 US academic and community hospitals from January 1, 2014, to December 31, 2015, who died in the hospital or were discharged to hospice and not readmitted. Medical records were reviewed from January 1, 2017, to March 31, 2018. Main Outcomes and Measures: Clinicians reviewed cases for sepsis during hospitalization using Sepsis-3 criteria, hospice-qualifying criteria on admission, immediate and underlying causes of death, and suboptimal sepsis-related care such as inappropriate or delayed antibiotics, inadequate source control, or other medical errors. The preventability of each sepsis-associated death was rated on a 6-point Likert scale. Results: The study cohort included 568 patients (289 [50.9%] men; mean [SD] age, 70.5 [16.1] years) who died in the hospital or were discharged to hospice. Sepsis was present in 300 hospitalizations (52.8%; 95% CI, 48.6%-57.0%) and was the immediate cause of death in 198 cases (34.9%; 95% CI, 30.9%-38.9%). The next most common immediate causes of death were progressive cancer (92 [16.2%]) and heart failure (39 [6.9%]). The most common underlying causes of death in patients with sepsis were solid cancer (63 of 300 [21.0%]), chronic heart disease (46 of 300 [15.3%]), hematologic cancer (31 of 300 [10.3%]), dementia (29 of 300 [9.7%]), and chronic lung disease (27 of 300 [9.0%]). Hospice-qualifying conditions were present on admission in 121 of 300 sepsis-associated deaths (40.3%; 95% CI 34.7%-46.1%), most commonly end-stage cancer. Suboptimal care, most commonly delays in antibiotics, was identified in 68 of 300 sepsis-associated deaths (22.7%). However, only 11 sepsis-associated deaths (3.7%) were judged definitely or moderately likely preventable; another 25 sepsis-associated deaths (8.3%) were considered possibly preventable. Conclusions and Relevance: In this cohort from 6 US hospitals, sepsis was the most common immediate cause of death. However, most underlying causes of death were related to severe chronic comorbidities and most sepsis-associated deaths were unlikely to be preventable through better hospital-based care. Further innovations in the prevention and care of underlying conditions may be necessary before a major reduction in sepsis-associated deaths can be achieved

Kinase insert domain receptor/vascular endothelial growth factor receptor 2 (KDR) genetic variation is associated with ovarian hyperstimulation syndrome

Background The objective of this investigation was to determine if kinase insert domain/vascular endothelial growth factor receptor 2 (KDR/VEGFR2) genetic variation was associated with the development of ovarian hyperstimulation syndrome (OHSS) in patients undergoing controlled ovarian hyperstimulation (COH). Methods This was a case-control study of 174 patients who underwent controlled ovarian stimulation. Patient blood samples were genotyped for single nucleotide polymorphisms (SNPs) spanning the KDR locus. OHSS development, clinical outcome variables, SNP and haplotype frequencies were compared between control (n = 155) and OHSS (n = 19) groups. Results Patients who developed OHSS had significantly higher response markers (estradiol levels of the day of hCG administration, number of follicles developed, number of eggs retrieved) than control patients. When adjusted for age and self-identified race, the rs2305945 G/T genotype was associated (P = 0.027) with a decreased risk (OR = 0.30; 95% CI = 0.10, 0.93) of developing OHSS using an overdominant model. The rs2305945 G/T variant was also associated with decreased COH response (number of follicles, number of eggs retrieved) in an overdominant model. The rs2305948, rs1870378, rs2305945 (C-T-G) haplotype was associated with both decreased COH response and OHSS risk (unadjusted OR = 0.10; 95% CI = 0.01, 0.80, P = 0.031). Conclusions The KDR receptor is believed to play a central role OHSS development and is a target for pharmacological prevention of OHSS. These results indicate that genetic variation in the KDR gene may impact individual risk of developing OHSS from COH. In addition, the rs2305948 SNP and C-T-G haplotype might serve as potential biomarkers for poor ovarian response to COH

Association between the luteinizing hormone/chorionic gonadotropin receptor (LHCGR) rs4073366 polymorphism and ovarian hyperstimulation syndrome during controlled ovarian hyperstimulation

Background The aim of this study was to determine the relationship between a purported luteinizing hormone/chorionic gonadotropin (LHCGR) high function polymorphism (rs4539842/insLQ) and outcome to controlled ovarian hyperstimulation (COH). Methods This was a prospective study of 172 patients undergoing COH at the Fertility and IVF Center at GWU. DNA was isolated from blood samples and a region encompassing the insLQ polymorphism was sequenced. We also investigated a polymorphism (rs4073366 G \u3e C) that was 142 bp frominsLQ. The association of the insLQ and rs4073366 alleles and outcome to COH (number of mature follicles, estradiol level on day of human chorionic gonadotropin (hCG) administration, the number of eggs retrieved and ovarian hyperstimulation syndrome (OHSS)) was determined. Results Increasing age and higher day 3 (basal) FSH levels were significantly associated with poorer response to COH. We found that both insLQ and rs4073366 were in linkage disequilibrium (LD) and no patients were homozygous for both recessive alleles (insLQ/insLQ; C/C). The insLQ variant was not significantly associated with any of the main outcomes to COH. Carrier status for the rs4073366 C variant was associated (P = 0.033) with an increased risk (OR 2.95, 95% CI = 1.09-7.96) of developing OHSS. Conclusions While age and day 3 FSH levels were predictive of outcome, we found no association betweeninsLQ and patient response to COH. Interestingly, rs4073366 C variant carrier status was associated with OHSS risk. To the best of our knowledge, this is the first report suggesting thatLHCGR genetic variation might function in patient risk for OHSS

Are atmospheric updrafts a key to unlocking climate forcing and sensitivity?

Both climate forcing and climate sensitivity persist as stubborn uncertainties limiting the extent to which climate models can provide actionable scientific scenarios for climate change. A key, explicit control on cloud–aerosol interactions, the largest uncertainty in climate forcing, is the vertical velocity of cloud-scale updrafts. Model-based studies of climate sensitivity indicate that convective entrainment, which is closely related to updraft speeds, is an important control on climate sensitivity. Updraft vertical velocities also drive many physical processes essential to numerical weather prediction. Vertical velocities and their role in atmospheric physical processes have been given very limited attention in models for climate and numerical weather prediction. The relevant physical scales range down to tens of meters and are thus frequently sub-grid and require parameterization. Many stateof- science convection parameterizations provide mass fluxes without specifying vertical velocities, and parameterizations that do provide vertical velocities have been subject to limited evaluation against what have until recently been scant observations. Atmospheric observations imply that the distribution of vertical velocities depends on the areas over which the vertical velocities are averaged. Distributions of vertical velocities in climate models may capture this behavior, but it has not been accounted for when parameterizing cloud and precipitation processes in current models. New observations of convective vertical velocities offer a potentially promising path toward developing process-level cloud models and parameterizations for climate and numerical weather prediction. Taking account of the scale dependence of resolved vertical velocities offers a path to matching cloud-scale physical processes and their driving dynamics more realistically, with a prospect of reduced uncertainty in both climate forcing and sensitivity

Complexities of chromium carcinogenesis: Role of cellular response, repair and recovery mechanisms

Certain hexavalent chromium (Cr(VI))-containing compounds are recognized occupational human lung carcinogens and may pose an environmental health risk. The carcinogenicity of Cr(VI) is targeted to particulate forms of moderate to low solubility. Soluble Cr(VI) oxyanions in the immediate cellular microenvironment traverse the cell membrane by non-specific anionic transporters. Cr(VI) is reductively metabolized within cells by agents including ascorbic acid (Asc), glutathione (GSH) and cysteine (Cys). During Cr(VI) reduction, a diverse range of genetic lesions are generated including Cr-DNA binary (mono) adducts, Cr-DNA ternary adducts, DNA protein crosslinks (DPCs), bi-functional (DNA interstrand crosslinks (ICLs)) adducts, single-strand breaks (SSBs) and oxidized bases. Some forms of Cr damage, such as ICLs, present physical barriers to DNA replication/transcription and, thus, likely promote a terminal cell fate such as apoptosis or terminal growth arrest. Other lesions, such as ternary DNA adducts, are potentially pre-mutagenic. Cr(VI) exposure elicits a classical DNA damage response within cells including activation of the p53 signaling pathway and cell cycle arrest or apoptosis. Moreover, Cr(VI) also induces the ATM-dependent DNA damage response pathway which is paradoxically required for both apoptosis and survival after Cr(VI) insult. In yeast, moderately cytotoxic concentrations of Cr(VI) result in an initial G1 arrest and delayed S phase progression, whereas less toxic levels of Cr(VI) induce G2 arrest, which requires homologous recombination for exit and survival. The past several years has witnessed many important advances in our understanding of the genetic/cellular damage produced by exposure to Cr(VI). Further information is needed regarding the potential involvement of oxygen radicals in Cr genotoxicity, the specific DNA repair pathways activated by Cr and the complex signaling mechanisms involved in the cellular response to Cr(VI). These pertinent issues must be considered in relation to the potential role that each plays in the induction of human respiratory tract cancer by particulate Cr(VI) compounds. © 2003 Elsevier B.V. All rights reserved

Improving nurse practitioners\u27 competence with genetics: Effectiveness of an online course.

© 2016 American Association of Nurse Practitioners. Purpose: The purpose of this study was to assess the effectiveness of an online genetics course for improving nurse practitioners\u27 knowledge, competence, and comfort with genetic principles and their application to clinical practice. Data sources: A genetics knowledge test and survey were administered to 232 nurse practitioner students, between 2011 and 2013, before and after completing a 15-week online genetics course taught by a multidisciplinary team of instructors at a private east coast U.S. university. The 65-item survey allowed participants to rate competence regarding genetic principles, diseases, and terminology, as well as comfort performing various clinical tasks related to genetics. The 21-item knowledge test contained multiple choice questions regarding core competencies in genetics. Paired t-tests were used to compare mean pre- and postscores. Conclusions: Participants significantly increased postcourse knowledge (p \u3c .001) and comfort with genetic core competencies and clinical skills related to genetics (p \u3c .001). This study demonstrates the effectiveness of an online genetics course for increasing nurse practitioners\u27 knowledge, competence, and confidence with genetics and identifies specific topics educators should consider when designing curricula for nurse practitioners. Implications for practice: Findings from this study can improve genetics education for nurse practitioners, which will in turn improve patient health